Targeted extracellular vesicles comprising membrane proteins with engineered glycosylation sites

ABSTRACT

Disclosed are extracellular vesicles comprising an engineered targeting protein for targeting the extracellular vesicles to target cells. The targeting protein is a fusion protein that includes a ligand, an engineered glycosylation site, and an exosome-targeting domain. Exemplary extracellular vesicles may include but are not limited to exosomes.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims the benefit of priority to U.S.Provisional Patent Application No. 62/233,625, filed on Sep. 28, 2015,the content of which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number P50CA090386 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

The field of the invention relates to the use of lipid particles fordelivering agents to target cells. In particular, the field of theinvention relates to secreted extracellular vesicles (EVs) that containa targeting membrane protein having an engineered glycosylation site.The secreted extracellular vesicles may be utilized to deliver an agentto a target cell, such as a therapeutic agent.

Secreted extracellular vesicles, such as exosomes, are nanometer-scalelipid vesicles that are produced by many cell types and transferproteins, nucleic acids, and other molecules between cells in the humanbody, as well as those of other animals. Targeted exosomes have a widevariety of potential therapeutic uses and have already been shown to beeffective for delivery of RNA to neural cells and tumor cells in mice.Here, we describe a method for displaying peptide-based targetingligands on the exterior of exosomes such that ligands are not degradedby endosomal proteases during exosome biogenesis. This method is novelin that it is the first to acknowledge and address the widespreadproblem of cleavage of targeting ligands from the luminal terminus extraof integral exosome membrane proteins during exosome biogenesis.Therefore this technology is the first robust method for display oftargeting ligands on the exterior of exosomes via the expression ofengineered proteins that localize to exosomes. This targeting system canbe used for engineering exosomes as targeted gene therapy or drugdelivery vehicles in vivo, which could be applied to a wide variety ofcell types and diseases.

SUMMARY

Disclosed are extracellular vesicles comprising an engineered targetingprotein that targets the extracellular vesicles to a target cell. Thetargeting protein is a fusion protein that includes as domains: aligand, an engineered glycosylation site, and an exosome-targetingdomain. Exemplary extracellular vesicles may include but are not limitedto exosomes.

The ligand of the fusion protein is expressed on the surface of theextracellular vesicles and targets the extracellular vesicles to targetcells. The engineered glycosylation site enables the fusion protein tobe glycosylated in the cell. Preferably, when the engineeredglycosylation site is glycosylated, the fusion protein and/or thecomponent domains of the fusion protein are protected from cleavage fromthe fusion protein and/or degradation in lysosomes. For example, whenthe engineered glycosylation site is glycosylated, preferably the ligandis protected from being cleaved from the fusion protein. Theexosome-targeting domain targets the fusion protein to intracellularvesicles such as lysosomes, where the fusion protein may be incorporatedinto the membranes of lysosomes and secreted as extracellular vesiclessuch as exosomes.

The extracellular vesicles further may comprise an agent, such as atherapeutic agent, and the extracellular vesicles may be utilized todeliver the comprised agent to a target cell. Agents comprised by theextracellular vesicles may include but are not limited to biologicalmolecules, such as cargo RNAs, and other small molecular therapeuticmolecules. For example, the fusion protein further may comprise anRNA-domain domain that binds to one or more RNA-motifs present on acargo RNA such that the fusion protein functions as a packaging proteinin order to package the cargo RNA into the extracellular vesicle, priorto the extracellular vesicles being secreted from a cell. In someembodiments, the packaging protein may be referred to as extracellularvesicle-loading protein or “EV-loading protein.”

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Exosome Production: Exosomes are formed when the intraluminalvesicles of a multivesicular body (MVB) are released during MVBbackfusion with the cell's outer membrane. Exosomes encapsulateendosomal membrane proteins, plasma membrane proteins, and cytoplasmicproteins and RNA.

FIG. 2. Exosome Delivery: Exosomes are taken up by recipient cells by avariety of mechanisms, and exosome cargo is delivered to the cytoplasmof the recipient cell, where it is functional.

FIG. 3. Schematic representation of one embodiment of fusion proteins ascontemplated herein. (Top) LAMP2b fusion proteins for expressing aprotein of interest on the exosome surface; (Bottom) LAMP2b fusionproteins for expressing a protein of interest on the exosome lumen.

FIG. 4. Schematic representation of one embodiment of a packagingprotein and cargo RNA as contemplated herein. The packaging protein is afusion protein comprising from N-terminus to C-terminus: a signalpeptide, at least a portion of LAMP2b comprising the exosome-targetingdomain, and at least a portion of the MS2 coat protein comprising theRNA-binding domain. The cargo RNA comprises the high affinity bindingloop of MS2 RNA fused at the 5′-terminus (top) or 3′-terminus (bottom)of a cargo sequence of interest.

FIG. 5. Packaging of cargo RNA comprising the MS2 RNA packaging signalinto exosomes in the presence of a fusion protein comprising the MS2coat protein RNA-binding domain

FIG. 6. Packaging of long cargo RNA into exosomes in the presence ofTAMEL packaging protein.

FIG. 7. Results of three experiments showing increased levels of dTomatocargo RNA in exosomes via CD63-MS2 active loading.

FIG. 8. Results of three experiments showing increased levels of dTomatocargo RNA in gesicles via VSVG-MS2 active loading.

FIG. 9. Stability of exosome-targeting peptides. A, this graphicillustrates the orientation of Lamp2b in the endosomal membrane andexposure of N-terminal peptides (N-term. peptide) to proteases. MVB,multivesicular body. C.-term., C terminus. B, transmission electronmicroscopy image of exosomes isolated by differential centrifugationfrom HEK293FT cell supernatant. C, size distribution of exosomessecreted by HEK293FT cells. D, enrichment of exosome-associated proteinCD63 in exosome preparations relative to β-actin. E and F, expression ofLamp2b fusion proteins in cell lysates (E) and exosomes (F), asevaluated by HA (C-terminal) and FLAG (N-terminal) Western blots.

FIG. 10. Glycosylation motif-mediated stabilization of Lamp2b fusionproteins. A, expression of Lamp2b fusion proteins including anengineered GNSTM glycosylation motif in cells. In this and subsequentfigures, the abbreviation “Xgs” is used to indicate a flexible linker Xamino acids in length, comprising glycine and serine residues. B,transfection efficiency of cells expressing Lamp2b fusion proteins.Error bars indicate mean±S.D. C and D, expression of Lamp2b fusionproteins in cell lysates (C) and exosomes (D) measured by HA(C-terminal) and FLAG (N-terminal) Western blots. E, cells were treatedwith either bafilomycin A1 (Baf), which blocks endosomal acidification,or leupeptin, which inhibits endosomal proteases.

FIG. 11. Impact of engineered glycosylation motif on targeting peptidebinding interactions. A and B, expression of FLAG-Lamp2b fusion proteinsin cell lysates (A) and exosomes (B) before and after pulldown withanti-FLAG beads. C, lysates from cells expressing NST-tagged FLAG-Lamp2bproteins were diluted 1:5 in TBS (where indicated) and pulled down toconfirm that apparent FLAG-mediated pulldown was not an artifact ofvariable levels of protein in the pulldown assay load. D, pulldown ofintact exosomes requires that the FLAG tag be expressed on the Lamp2b Nterminus (exosome exterior).

FIG. 12. Glycosylation-enhanced targeted delivery of exosomes toneuroblastoma cells. A, equivalent numbers of PHK67-labeled exosomeswere incubated with Neuro2A cells for 2 h (˜3×10⁹ exosomes per 1×10⁵cells), and uptake was quantified by flow cytometry. The shadedhistogram is the exosome-free negative control to evaluate excess dyeand dye-derived micelles. exos, exosomes. B, quantification of peakspresented in panel A.

DETAILED DESCRIPTION

The present invention is described herein using several definitions, asset forth below and throughout the application.

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, “a fusion protein,” “an RNA,”and “a loop” should be interpreted to mean “one or more fusionproteins,” “one or more RNAs,” and “one or more loops,” respectively. An“engineered glycosylation site” should be interpreted to mean “one ormore engineered glycosylation sites.”

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of these terms which are not clear to persons ofordinary skill in the art given the context in which they are used,“about” and “approximately” will mean plus or minus ≤10% of theparticular term and “substantially” and “significantly” will mean plusor minus >10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising” in that these latterterms are “open” transitional terms that do not limit claims only to therecited elements succeeding these transitional terms. The term“consisting of,” while encompassed by the term “comprising,” should beinterpreted as a “closed” transitional term that limits claims only tothe recited elements succeeding this transitional term. The term“consisting essentially of,” while encompassed by the term “comprising,”should be interpreted as a “partially closed” transitional term whichpermits additional elements succeeding this transitional term, but onlyif those additional elements do not materially affect the basic andnovel characteristics of the claim.

Disclosed are extracellular vesicles comprising a targeting protein thattargets the extracellular vesicles to a target cell. Exemplaryextracellular vesicles may include but are not limited to exosomes.However, the term “extracellular vesicles” should be interpreted toinclude all nanometer-scale lipid vesicles that are secreted by cellssuch as secreted vesicles formed from lysosomes.

The disclosed extracellular vesicles comprise a “targeting protein.” Thetarget protein may be described as a “fusion protein,” and the term“targeting protein” and “fusion protein” may be used interchangeablyherein depending on context. The fusion protein typically includes: (i)a ligand (e.g., a heterologous ligand) that is expressed on the surfaceof the extracellular vesicles and targets the extracellular vesicles totarget cells, (ii) an engineered glycosylation site (e.g., aheterologous glycosylation site), and (iii) an exosome-targeting domain.In some embodiments, the fusion protein has a luminal N-terminus and acytosolic C-terminus and the fusion protein comprises from N-terminus toC-terminus: the ligand, the glycosylation site, and theexosome-targeting domain.

The ligand of the fusion protein typically is a heterologous amino acidsequence (i.e., relative to the engineered glycosylation site and/orrelative to the exosome-targeting domain) that binds to a receptorpresent on the surface of a target cell (e.g., a protein receptor, acarbohydrate receptor, or a lipid receptor present on the surface of acell). For example, suitable ligands may include a ligand for a cellreceptor present on a target cell, or an antibody or binding fragmentthereof that binds to a cell receptor or other membrane protein presenton a target cell. The ligand of the fusion protein typically is presentat the luminal end of the fusion protein, which optionally may be theN-terminus of the fusion protein. For example, the fusion protein maycomprise a structure as follows: N_(ter)-signal peptide-ligand fortarget cell—engineered glycosylation site—exosome targetingdomain—C_(ter).

The engineered glycosylation site of the fusion protein may be definedas a sequence of amino acids that is a target for enzymatic, N-linkedglycosylation when the fusion protein is expressed in a cell. Theengineered glycosylation site may be present adjacent to the ligand ofthe fusion protein (e.g., N_(ter)-signal peptide-ligand for targetcell-engineered glycosylation site-exosome targeting domain—C_(ter)).Preferably, when the engineered glycosylation site is glycosylated, thefusion protein or the component domains of the fusion protein areprotected from cleavage from the fusion protein and/or degradation inlysosomes. (See Hung et al.; and Schulz). For example, the fusionprotein may include a glycosylation motif and/or may be engineered toinclude a glycosylation motif in order to protect or inhibit the fusionprotein and/or component domains of the fusion protein from proteolyticcleavage from the fusion protein or degradation, such as intracellularproteolysis. (See Kundra et al.). Suitable glycosylation motifs mayinclude the NX(S/T) consensus sequon and in particular the NST sequon(SEQ ID NO:37). In some embodiments, the fusion protein may include aGNSTM sequon (SEQ ID NO:38). The NST sequence is a known N-linkedglycosylation sequon, and the amino acids G and M flanking the sequonmay increase glycosylation frequency in mammals. (See Ballo-Polo etal.). The glycosylation site typically is “engineered,” meaning that theglycosylation site typically is not naturally present in the fusionprotein or any of the component proteins of the fusion protein, andrather, is introduced into the fusion protein, for example, byrecombinant engineering.

The exosome targeting domain of the fusion protein may include but isnot limited to a domain of an exosomal-associated protein and/or alysosome-associated protein. A database of exosomal proteins, RNA, andlipids is provided by ExoCarta at its website. (See also, Mathivanan etal., Nucl. Acids Res. 2012, Vol. 40, Database issue D1241-1244,published online 11 Oct. 2011, the content of which is incorporatedherein by reference in its entirety.) Suitable exosomal-associatedproteins, which also may be described as exosomal vesicle-enrichedproteins or (EEPs) have been described. (See Hung and Leonard, “Aplatform for actively loading cargo RNA to elucidate limiting steps inEV-mediated delivery,” J. Extracellular Vesicles, 2016, 5: 31027,published 13 May 2016, the content of which is incorporated herein byreference in its entirety). In some embodiments, suitable domains oflysosome-associated proteins may include domains from lysosome membraneproteins having a luminal N-terminus and a cytoplasmic C-terminus,although membrane proteins having different orientations also may besuitable (e.g. membrane proteins having a luminal C-terminus and acytoplasmic N-terminus).

In some embodiments, the exosome-targeting domain is a domain of alysosome-associated protein. Suitable lysosome-associated protein mayinclude, but are not limited to, lysosome membrane proteins. (SeeSaftig, Lysosomes, Chapter 6, “Lysosome Membrane Proteins” 2004).Lysosome-associated membrane proteins (LAMPs) and lysosome integralmembrane proteins (LIMPs) are the most abundant proteins of the lysosomemembrane. (See id.).

In some embodiments of the fusion proteins disclosed herein, theexosome-targeting domain is an exosome-targeting domain of a LAMP.Suitable LAMPs may include, but are not limited to, LAMP-1 and LAMP-2,and isoforms thereof. (See Fukuda et al., “Cloning of cDNAs EncodingHuman Lysosomal Membrane Glycoproteins, h-lamp-1 and h-lamp-2,” J. Biol.Chem., Vol. 263, No. 35 Dec. 1988, pp. 18920-18928; and Fukuda,“Lysosomal Membrane Glycoproteins,” J. Biol. Chem., Vol. 266, No. 32,November 1991, pp. 21327, 21330.) LAMPs are lysosome-membrane proteinshaving a luminal (i.e., extracytoplasmic)N-terminus and a cytoplasmicC-terminus. (See id.). The mRNAs for expressing LAMPs may be processeddifferently to give isoforms. For example, there are three isoforms forLAMP-2 designated as LAMP-2a, LAMP-2b, and LAMP-2c. (See UniProtDatabase, entry number P13473-LAMP2_HUMAN, the contents of which isincorporated herein by reference in its entirety). LAMP-1 has a singleisoform. (See UniProt Database, entry number P11279-LAMP1_HUMAN, thecontent of which is incorporated herein by reference in its entirety).The full-length amino acid sequence of LAMP-2a, LAMP-2b, and LAMP-2c areprovided herein as SEQ ID NOs:20, 21, and 22, respectively. Thefull-length amino acid sequence of LAMP-1 is provided herein as SEQ IDNO:26. The fusion proteins disclosed herein may include the full-lengthamino acid sequence of a LAMP or a variant thereof as contemplatedherein having a percentage of sequence identity in comparison to theamino acid sequence of the wild-type LAMP, or a fragment thereofcomprising a portion of the wild-type LAMP (e.g., SEQ ID NOs:23, 24, 25,and 27 comprising a portion of the C-termini of LAMP-2a, LAMP-2b,LAMP-2c, and LAMP-1, respectively).

For LAMPs, the C-terminus (e.g., comprising the 10-11 C-terminal aminoacids) has been shown to be important for targeting LAMPs to lysosomes.(See id.; and Fukuda 1991). In some embodiments of the disclosedextracellular vesicles, the fusion protein comprises the RNA-bindingdomain fused to the C-terminus of one of SEQ ID NOs:23, 24, 25, and 27,which comprise a portion of the C-termini of LAMP-2a, LAMP-2b, LAMP-2c,and LAMP-1, respectively). The fusion protein may include thecytoplasmic domain of a LAMP and optionally may include additional aminoacid sequences (e.g., at least a portion of the transmembrane domainand/or at least a portion of the luminal domain).

In some embodiments, the exosome-targeting domain is anexosome-targeting domain of a LIMP. Suitable LIMPs may include, but arenot limited to, LIMP-1 (CD63) and LAMP-2, and isoforms thereof. LIMPsare lysosome-membrane proteins having one or more luminal domains,multiple transmembrane domains, and a cytoplasmic C-terminus. (See Ogataet al., “Lysosomal Targeting of Limp II Membrane Glycoprotein Requires aNovel Leu-Ile Motif at a Particular Position in Its Cytoplasmic Tail,”J. Biol. Chem., Vol. 269, No. 7, February 1994, pp. 5210-5217). ThemRNAs for expressing LIMPs may be processed differently to giveisoforms. For example, there are three isoforms for LIMP-1 designated asLIMP-1a, LIMP-1b, and LIMP-1c and two isoforms for LIMP-2 designated asLIMP-2a and LIMP-2b. (See UniProt Database, entry numberQ10148-SCRB2_HUMAN, and UniProt Database, entry numberP08962-CD63_HUMAN, the content of which is incorporated herein byreference in its entirety). The full-length amino acid sequence ofLIMP-1a, LIMP-1b, and LIMP-1c are provided herein as SEQ ID NOs:28, 29,and 30, respectively. The full-length amino acid sequence of LIMP-2A andLIMP-2b are provided herein as SEQ ID NOs:32 and 33, respectively. Thefusion proteins disclosed herein may include the full-length amino acidsequence of a LIMP or a variant thereof as contemplated herein having apercentage of sequence identity in comparison to the amino acid sequenceof the wild-type LIMP, or a fragment thereof comprising a portion of thewild-type LIMP (e.g., SEQ ID NO:31 comprising a portion of the C-terminiof LIMP-1a, LIMP-1b, LIMP-1C and SEQ ID NO:34 comprising a portion ofthe C-termini of LIMP-2a and LIMP-2b).

For LIMPs, the C-terminus (e.g., comprising the 14-19 C-terminal aminoacids) has been shown to be important for targeting LAMPs to lysosomes.(See Ogata et al.). In some embodiments of the disclosed extracellularvesicles, the fusion protein comprises the RNA-binding domain fused tothe C-terminus of one of SEQ ID NOs:31 and 34, which comprise a portionof the C-termini of LIMP-1a, LIMP-1b, LIMP-1c, and LIMP-2a and LIMP-2b).The fusion protein may include the cytoplasmic domain of a LIMP andoptionally may include additional amino acid sequences (e.g., at least aportion of the transmembrane domain and/or at least a portion of theluminal domain).

In some embodiments of the fusion proteins disclosed herein theexosome-targeting domain is an exosome-targeting domain of CD63 orisoforms thereof. The CD63 protein alternately may be referred to byaliases including Lysosome-Integrated Membrane Protein 1 (LIMP-1), MLA1,Lysosomal-Associated Membrane Protein 3, Ocular Melanoma-AssociatedAntigen, Melanoma 1 Antigen, Melanoma-Associated Antigen ME491,Tetraspanin-30, Granulophysin, and Tspan-30. Isoforms of CD63 mayinclude CD63 Isoform A (i.e., LIMP-1a (SEQ ID NO:28)), CD63 Isoform C(i.e., LIMP-1b (SEQ ID NO:29)) and CD63 Isoform D Precursor (providedherein as SEQ ID NO:35).

In some embodiments of the fusion proteins disclosed herein theexosome-targeting domain is an exosome-targeting domain of a viraltransmembrane protein. Viral transmembrane proteins are known in theart. (See e.g., Fields Virology, Sixth Edition, 2013. See also White etal., Crit. Rev. Biochem. Mol. Biol. 2008; 43(3): 189-219). Specifically,the exosome-targeting domain may be an exosome-targeting domain of the Gglycoprotein of Vesicular Stomatitis Virus (VSV G-protein). The aminoacid sequence of VSV G-protein is provided herein as SEQ ID NO:36.

The disclosed extracellular vesicles further may comprise an agent, suchas a therapeutic agent, where the extracellular vesicles deliver theagent to a target cell. Agents comprised by the extracellular vesiclesmay include but are not limited to therapeutic drugs (e.g., smallmolecule drugs), therapeutic proteins, and therapeutic nucleic acids(e.g., therapeutic RNA). In some embodiments, the disclosedextracellular vesicles comprise a therapeutic RNA as a so-called “cargoRNA.” For example, in some embodiments the fusion protein further maycomprise an RNA-domain (e.g., at a cytosolic C-terminus of the fusionprotein) that binds to one or more RNA-motifs present in the cargo RNAin order to package the cargo RNA into the extracellular vesicle, priorto the extracellular vesicles being secreted from a cell. As such, thefusion protein may function as both of a “targeting protein” and a“packaging protein.” In some embodiments, the packaging protein may bereferred to as extracellular vesicle-loading protein or “EV-loadingprotein.” (See Hung and Leonard, “A platform for actively loading cargoRNA to elucidate limiting steps in EV-mediated delivery,” J.Extracellular Vesicles, 2016, 5: 31027, published 13 May 2016, thecontent of which is incorporated herein by reference in its entirety.)

In embodiments in which the extracellular vesicles comprise a cargo RNA,the cargo RNA which may be described as a fusion RNA comprising: (1) aRNA-motif that binds the RNA-binding domain of the fusion protein andfurther, (2) additional functional RNA sequences that be utilized fortherapeutic purposes (e.g., miRNA, shRNA, mRNA, ncRNA, sgRNA or acombination of any of these RNAs).

The cargo RNA of the disclosed extracellular vesicles may be of anysuitable length. For example, in some embodiments the cargo RNA may havea nucleotide length of at least about 10 nt, 20 nt, 30 nt, 40 nt, 50 nt,100 nt, 200 nt, 500 nt, 1000 nt, 2000 nt, 5000 nt, or longer. In otherembodiments, the cargo RNA may have a nucleotide length of no more thanabout 5000 nt, 2000 nt, 1000 nt, 500 nt, 200 nt, 100 nt, 50 nt, 40 nt,30 nt, 20 nt, or 10 nt. In even further embodiments, the cargo RNA mayhave a nucleotide length within a range of these contemplated nucleotidelengths, for example, a nucleotide length between a range of about 10nt-5000 nt, or other ranges. The cargo RNA of the disclosedextracellular vesicles may be relatively long, for example, where thecargo RNA comprises an mRNA or another relatively long RNA.

Suitable RNA-binding domains and RNA-motifs for the components of thepresently disclosed extracellular vesicles may include, but are notlimited to, RNA-binding domains and RNA-motifs of bacteriophage. (See,e.g., Keryer-Bibens et al., “Tethering of proteins to RNAs bybacteriophage proteins,” Biol. Cell (2008) 100, 125-138, the content ofwhich is incorporated herein by reference in its entirety).

In some embodiments of the disclosed extracellular vesicles, theRNA-binding domain of the fusion protein is an RNA-binding domain ofcoat protein of MS2 bacteriophage or R17 bacteriophage, which may beconsidered to be interchangeable. (See, e.g., Keryer-Bibens et al.; andStockley et al., “Probing sequence-specific RNA recognition by thebacteriophage MS2 coat protein,” Nucl. Acids. Res., 1995, Vol. 23, No.13, pages 2512-2518, the content of which is incorporated herein byreference in its entirety). The full-length amino acid sequence of thecoat protein of MS2 bacteriophage is provided herein as SEQ ID NO:1. Thefusion proteins disclosed herein may include the full-length amino acidsequence of the coat protein of MS2 bacteriophage or a variant thereofas contemplated herein having a percentage of sequence identity incomparison to the amino acid sequence of the coat protein of MS2bacteriophage, or a fragment thereof comprising a portion of the coatprotein of MS2 bacteriophage (e.g., the RNA-binding domain of MS2 or SEQID NO:2, comprising the amino acid sequence (2-22) of the coat proteinof MS2 bacteriophage).

In embodiments where the fusion protein comprises an RNA-binding domainof coat protein of MS2 bacteriophage, the cargo RNA typically comprisesan RNA-motif of MS2 bacteriophage RNA which may form a high affinitybinding loop that binds to the RNA-binding domain of the fusion protein.(See Peabody et al., “The RNA binding site of bacteriophage MS2 coatprotein,” The EMBO J., vol. 12, no. 2, pp. 595-600, 1993; Keryer-Bibenset al.; and Stockley et al., the contents of which are incorporatedherein by reference in their entireties). The RNA-motif of MS2bacteriophage and R17 bacteriophage has been characterized. (See id.).The RNA-motif has been determined to comprise minimally a 21-ntstem-loop structure where the identity of the nucleotides forming thestem do not appear to influence the affinity of the coat protein for theRNA-motif, but where the sequence of the loop contains a 4-nt sequence(AUUA (SEQ ID NO:3)), which does influence the affinity of the coatprotein for the RNA-motif. Also important, is an unpaired adenosine twonucleotides upstream of the loop. In some embodiments of the disclosedextracellular vesicles, the RNA-motif comprises one or more highaffinity binding loops comprising a sequence and structure selected fromthe group consisting of:

where N—N is any two base-paired RNA nucleotides (e.g., where eachoccurrence of N—N is independently selected from any of A-U, C-G, G-C,G-U, U-A, or U-G, and each occurrence of N—N may be the same ordifferent). Specifically, the high affinity binding loop may comprise asequence selected from the group consisting of SEQ ID NO:7(5′-ACAUGAGGAUUACCCAUGU-3′), SEQ ID NO:8 (5′-ACAUGAGGACUACCCAUGU-3′),and SEQ ID NO:9 (5′-ACAUGAGGAUCACCCAUGU-3′), or a variant thereof havinga percentage sequence identity.

Preferably, the RNA-binding domain of the fusion protein binds to theRNA-motif with an affinity of at least about 1×10⁻⁸ M. More preferably,the RNA-binding domain of the fusion protein binds to the RNA-motif withan affinity of at least about 1×10⁻⁹ M, even more preferably with anaffinity of at least about 1×10⁻¹⁰ M.

In addition to the RNA-motif for binding to the RNA-binding domain ofthe fusion protein, the cargo RNA may include additional functional RNAsequences that be utilized for therapeutic purposes (e.g., miRNA, shRNA,mRNA, ncRNA, sgRNA, or a combination of any of these RNAs). (See Marcuset al., “FedExosomes: Engineering Therapeutic Biological Nanoparticlesthat Truly Deliver,” Pharmaceuticals 2013, 6, 659-680; György et al.,Therapeutic application of extracellular vesicles: clinical promise andopen questions,” Annu. Rev. Pharmacol. Toxicol. 2015; 55:439-64, Epub2014 Oct. 3, the contents of which are incorporated herein by referencein their entireties). As such, the cargo RNA may be characterized as ahybrid RNA including the RNA-motif for binding to the RNA-binding domainof the fusion protein and including an additional RNA (e.g., miRNA,shRNA, mRNA, ncRNA, sgRNA, or a combination of any of these RNAs fusedat the 5′-terminus or 3′-terminus or at an internal portion within theRNA), which may be a therapeutic RNA.

In other embodiments of the disclosed extracellular vesicles, theRNA-binding domain of the fusion protein is an RNA-binding domain of theN-protein of a lambdoid bacteriophage, which may include but is notlimited to lambda bacteriophage, P22 bacteriophage, and phi21bacteriophage. (See, e.g., Keryer-Bibens et al.; Bahadur et al.,“Binding of the Bacteriophage P22 N-peptide to the boxB RNA-motifStudied by Molecule Dynamics Simulations,” Biophysical J., Vol., 97,December 2009, 3139-3149; Cilley et al., “Structural mimicry in thephage phi21 N peptide-boxB RNA complex,” RNA (2003), 9:663-376; thecontents of which are incorporated herein by reference in theirentireties). The full-length amino acid sequence of the N-protein oflambda bacteriophage, P22 bacteriophage, and phi21 bacteriophage areprovided herein as SEQ ID NOs:10, 11, and 12, respectively. The fusionproteins disclosed herein may include the full-length amino acidsequence of the N-protein of the lambdoid bacteriophage or a variantthereof as contemplated herein having a percentage of sequence identityin comparison to the amino acid sequence of the N-protein of thelambdoid bacteriophage, or a fragment thereof comprising a portion ofthe N-protein of the lambdoid bacteriophage (e.g., the RNA-bindingdomain of the N-protein of any of lambda bacteriophage, P22bacteriophage, and phi21 bacteriophage, or SEQ ID NOs:13, 14, and 15,comprising portions of the N-proteins of lambda bacteriophage, P22bacteriophage, and phi21 bacteriophage, respectively).

In embodiments where the fusion protein comprises an RNA-binding domainof coat protein of a lambdoid bacteriophage, the cargo RNA typicallycomprises an RNA-motif of lambda bacteriophage RNA which may form a highaffinity binding loop called “boxB” that binds to the RNA-binding domainof the fusion protein. (See Keryer-Bibens et al.). BoxB of lambdoidbacteriophage has been characterized. (See id.; Bahadur, et al.; andCilley et al.). For lambda bacteriophage, boxB has been determined tocomprise minimally a 15-nt stem-loop structure where the identity of thenucleotides forming the stem and loop influence the affinity of the coatprotein for the RNA-motif. (See Keryer-Bibens et al.). In someembodiments of the disclosed extracellular vesicles, the RNA-motifcomprises one or more high affinity binding loops comprising a sequenceand structure selected from the group consisting of:

or a variant thereof having a percentage sequence identity, where thevariant binds to the RNA-binding domain of the fusion protein.Preferably, the RNA-motif binds to the RNA-binding domain of the fusionprotein with an affinity of at least about 1×10⁻⁸ M, more preferablywith an affinity of at least about 1×10⁻⁹ M, even more preferably withan affinity of at least about 1×10⁻¹⁹ M.

For P22 bacteriophage, boxB has been determined to comprise minimally a15-nt stem-loop structure where the identity of the nucleotides formingthe stem and loop influence the affinity of the coat protein for theRNA-motif. (See Bahadur et al.). In some embodiments of the disclosedextracellular vesicles, the RNA-motif comprises one or more highaffinity binding loops comprising a sequence and structure of:

For phi21 bacteriophage, boxB has been determined to comprise minimallya 20-nt stem-loop structure where the identity of the nucleotidesforming the stem and loop influence the affinity of the coat protein forthe RNA-motif. (See Cilley et al.). In some embodiments of the disclosedextracellular vesicles, the RNA-motif comprises one or more highaffinity binding loops comprising a sequence and structure of:

The disclosed extracellular vesicles may be prepared by methods known inthe art. For example, the disclosed extracellular vesicles may beprepared by expressing in a eukaryotic cell (a) an mRNA that encodes thepackaging/fusion protein and (b) expressing in the eukaryotic cell thecargo RNA (or transducing the eukaryotic cell with the cargo RNA thathas been prepared in silico). The mRNA for the packaging/fusion proteinand the cargo RNA may be expressed from vectors that are transfectedinto suitable production cells for producing the disclosed extracellularvesicles. The mRNA for the packaging/fusion protein and the cargo RNAmay be expressed from the same vector (e.g., where the vector expressesthe mRNA for the packaging/fusion protein and the cargo RNA fromseparate promoters), or the mRNA for the packaging/fusion protein andthe cargo RNA may be expressed from separate vectors. The vector orvectors for expressing the mRNA for the packaging/fusion protein and thecargo RNA may be packaged in a kit designed for preparing the disclosedextracellular vesicles.

Also contemplated herein are methods for using the disclosedextracellular vesicles. For example, the disclosed extracellularvesicles may be used for delivering a therapeutic agent such as cargoRNA to a target cell, where the methods include contacting the targetcell with the disclosed extracellular vesicles. The disclosedextracellular vesicles may be formulated as part of a pharmaceuticalcomposition for treating a disease or disorder and the pharmaceuticalcomposition may be administered to a patient in need thereof to deliverythe cargo RNA to target cells in order to treat the disease or disorder.

The disclosed extracellular vesicles may comprise novel proteins,polypeptides, or peptides. As used herein, the terms “protein” or“polypeptide” or “peptide” may be used interchangeable to refer to apolymer of amino acids. Typically, a “polypeptide” or “protein” isdefined as a longer polymer of amino acids, of a length typically ofgreater than 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” isdefined as a short polymer of amino acids, of a length typically of 50,40, 30, 20 or less amino acids.

A “protein” as contemplated herein typically comprises a polymer ofnaturally or non-naturally occurring amino acids (e.g., alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline). The proteins contemplated herein may be further modified invitro or in vivo to include non-amino acid moieties. These modificationsmay include but are not limited to acylation (e.g., O-acylation(esters), N-acylation (amides), S-acylation (thioesters)), acetylation(e.g., the addition of an acetyl group, either at the N-terminus of theprotein or at lysine residues), formylation lipoylation (e.g.,attachment of a lipoate, a C8 functional group), myristoylation (e.g.,attachment of myristate, a C14 saturated acid), palmitoylation (e.g.,attachment of palmitate, a C16 saturated acid), alkylation (e.g., theaddition of an alkyl group, such as an methyl at a lysine or arginineresidue), isoprenylation or prenylation (e.g., the addition of anisoprenoid group such as farnesol or geranylgeraniol), amidation atC-terminus, glycosylation (e.g., the addition of a glycosyl group toeither asparagine, hydroxylysine, serine, or threonine, resulting in aglycoprotein). Distinct from glycation, which is regarded as anonenzymatic attachment of sugars, polysialylation (e.g., the additionof polysialic acid), glypiation (e.g., glycosylphosphatidylinositol(GPI) anchor formation, hydroxylation, iodination (e.g., of thyroidhormones), and phosphorylation (e.g., the addition of a phosphate group,usually to serine, tyrosine, threonine or histidine).

The term “amino acid residue” also may include amino acid residuescontained in the group consisting of homocysteine, 2-Aminoadipic acid,N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, β-alanine,β-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid,3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinicacid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid,allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine,3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid,6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine,Norvaline, 2,2′-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionicacid, Ornithine, and N-Ethylglycine.

The proteins disclosed herein may include “wild type” proteins andvariants, mutants, and derivatives thereof. As used herein the term“wild type” is a term of the art understood by skilled persons and meansthe typical form of an organism, strain, gene or characteristic as itoccurs in nature as distinguished from mutant or variant forms. As usedherein, a “variant, “mutant,” or “derivative” refers to a proteinmolecule having an amino acid sequence that differs from a referenceprotein or polypeptide molecule. A variant or mutant may have one ormore insertions, deletions, or substitutions of an amino acid residuerelative to a reference molecule. A variant or mutant may include afragment of a reference molecule. For example, a mutant or variantmolecule may one or more insertions, deletions, or substitution of atleast one amino acid residue relative to a reference polypeptide (e.g.,any of SEQ ID NOs: 1, 2, 10-15, and 20-36). The sequence of thefull-length coat protein of MS2 bacteriophage, the sequence of thefull-length N-protein of lambda bacteriophage, the sequence of thefull-length N-protein of P22 bacteriophage, the sequence of thefull-length N-protein of phi21 bacteriophage, the sequence of thefull-length LAMP-2a, the sequence of the full-length LAMP-2b, and thesequence of the full-length LAMP-2c, are presented as SEQ ID NOs:1, 10,11, 12, 20, 21, and 22, respectively, and may be used as a reference inthis regard.

Regarding proteins, a “deletion” refers to a change in the amino acidsequence that results in the absence of one or more amino acid residues.A deletion may remove at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, ormore amino acids residues. A deletion may include an internal deletionand/or a terminal deletion (e.g., an N-terminal truncation, a C-terminaltruncation or both of a reference polypeptide). A “variant,” “mutant,”or “derivative” of a reference polypeptide sequence may include adeletion relative to the reference polypeptide sequence.

Regarding proteins, “fragment” is a portion of an amino acid sequencewhich is identical in sequence to but shorter in length than a referencesequence. A fragment may comprise up to the entire length of thereference sequence, minus at least one amino acid residue. For example,a fragment may comprise from 5 to 1000 contiguous amino acid residues ofa reference polypeptide, respectively. In some embodiments, a fragmentmay comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90,100, 150, 250, or 500 contiguous amino acid residues of a referencepolypeptide. Fragments may be preferentially selected from certainregions of a molecule. The term “at least a fragment” encompasses thefull length polypeptide. For example, a fragment of a protein maycomprise or consist essentially of a contiguous portion of an amino acidsequence of the full-length proteins of any of SEQ ID NOS: 1, 2, 10-15,and 20-36. A fragment may include an N-terminal truncation, a C-terminaltruncation, or both truncations relative to the full-length protein. A“variant,” “mutant,” or “derivative” of a reference polypeptide sequencemay include a fragment of the reference polypeptide sequence.

Regarding proteins, the words “insertion” and “addition” refer tochanges in an amino acid sequence resulting in the addition of one ormore amino acid residues. An insertion or addition may refer to 1, 2, 3,4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or more aminoacid residues. A “variant,” “mutant,” or “derivative” of a referencepolypeptide sequence may include an insertion or addition relative tothe reference polypeptide sequence. A variant of a protein may haveN-terminal insertions, C-terminal insertions, internal insertions, orany combination of N-terminal insertions, C-terminal insertions, andinternal insertions.

Regarding proteins, the phrases “percent identity” and “% identity,”refer to the percentage of residue matches between at least two aminoacid sequences aligned using a standardized algorithm. Methods of aminoacid sequence alignment are well-known. Some alignment methods take intoaccount conservative amino acid substitutions. Such conservativesubstitutions, explained in more detail below, generally preserve thecharge and hydrophobicity at the site of substitution, thus preservingthe structure (and therefore function) of the polypeptide. Percentidentity for amino acid sequences may be determined as understood in theart. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated hereinby reference in its entirety). A suite of commonly used and freelyavailable sequence comparison algorithms is provided by the NationalCenter for Biotechnology Information (NCBI) Basic Local Alignment SearchTool (BLAST), which is available from several sources, including theNCBI, Bethesda, Md., at its website. The BLAST software suite includesvarious sequence analysis programs including “blastp,” that is used toalign a known amino acid sequence with other amino acids sequences froma variety of databases. As described herein, variants, mutants, orfragments (e.g., a protein variant, mutant, or fragment thereof) mayhave 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%,50%, 40%, 30%, or 20% amino acid sequence identity relative to areference molecule (e.g., relative to any of SEQ ID NOs: 1, 2, 10-15,and 20-36).

Regarding proteins, percent identity may be measured over the length ofan entire defined polypeptide sequence, for example, as defined by aparticular SEQ ID number, or may be measured over a shorter length, forexample, over the length of a fragment taken from a larger, definedpolypeptide sequence, for instance, a fragment of at least 15, at least20, at least 30, at least 40, at least 50, at least 70 or at least 150contiguous residues. Such lengths are exemplary only, and it isunderstood that any fragment length supported by the sequences shownherein, in the tables, figures or Sequence Listing, may be used todescribe a length over which percentage identity may be measured.

Regarding proteins, the amino acid sequences of variants, mutants, orderivatives as contemplated herein may include conservative amino acidsubstitutions relative to a reference amino acid sequence. For example,a variant, mutant, or derivative protein may include conservative aminoacid substitutions relative to a reference molecule. “Conservative aminoacid substitutions” are those substitutions that are a substitution ofan amino acid for a different amino acid where the substitution ispredicted to interfere least with the properties of the referencepolypeptide. In other words, conservative amino acid substitutionssubstantially conserve the structure and the function of the referencepolypeptide. The following table provides a list of exemplaryconservative amino acid substitutions which are contemplated herein:

Original Conservative Residue Substitution Ala Gly, Ser Arg His, Lys AsnAsp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln,His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg,Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser,Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, ThrConservative amino acid substitutions generally maintain (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a beta sheet or alpha helical conformation, (b) thecharge or hydrophobicity of the molecule at the site of thesubstitution, and/or (c) the bulk of the side chain.

The disclosed proteins, mutants, variants, or described herein may haveone or more functional or biological activities exhibited by a referencepolypeptide (e.g., one or more functional or biological activitiesexhibited by wild-type protein). For example, the disclosed proteins,mutants, variants, or derivatives thereof may have one or morebiological activities that include binding to a single-stranded RNA,binding to a double-stranded RNA, binding to a target polynucleotidesequence, and targeting a protein to a vesicle (e.g. a lysosome orexosome).

The disclosed proteins may be substantially isolated or purified. Theterm “substantially isolated or purified” refers to proteins that areremoved from their natural environment, and are at least 60% free,preferably at least 75% free, and more preferably at least 90% free,even more preferably at least 95% free from other components with whichthey are naturally associated.

Also disclosed herein are polynucleotides, for example polynucleotidesequences that encode proteins (e.g., DNA that encodes a polypeptidehaving the amino acid sequence of any of SEQ ID NOs: 1, 2, 10-15, and20-36 or a polypeptide variant having an amino acid sequence with atleast about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,or 99% sequence identity to any of SEQ ID NOs: 1, 2, 10-15, and 20-36;DNA encoding the polynucleotide sequence of any of SEQ ID NOs:3-9 and16-19 or encoding a polynucleotide variant having a nucleotide sequencewith at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, or 99% sequence identity to any of SEQ ID NOs:3-9 and 16-19;RNA comprising the polynucleotide sequence of any of SEQ ID NOs:3-9 and16-19 or a polynucleotide variant having a nucleotide sequence with atleast about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,or 99% sequence identity to any of SEQ ID NOs:3-9 and 16-19).

The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid”and “nucleic acid sequence” refer to a nucleotide, oligonucleotide,polynucleotide (which terms may be used interchangeably), or anyfragment thereof. These phrases also refer to DNA or RNA of genomic,natural, or synthetic origin (which may be single-stranded ordouble-stranded and may represent the sense or the antisense strand).

Regarding polynucleotide sequences, the terms “percent identity” and “%identity” refer to the percentage of residue matches between at leasttwo polynucleotide sequences aligned using a standardized algorithm.Such an algorithm may insert, in a standardized and reproducible way,gaps in the sequences being compared in order to optimize alignmentbetween two sequences, and therefore achieve a more meaningfulcomparison of the two sequences. Percent identity for a nucleic acidsequence may be determined as understood in the art. (See, e.g., U.S.Pat. No. 7,396,664, which is incorporated herein by reference in itsentirety). A suite of commonly used and freely available sequencecomparison algorithms is provided by the National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST), which is available from several sources, including the NCBI,Bethesda, Md., at its website. The BLAST software suite includes varioussequence analysis programs including “blastn,” that is used to align aknown polynucleotide sequence with other polynucleotide sequences from avariety of databases. Also available is a tool called “BLAST 2Sequences” that is used for direct pairwise comparison of two nucleotidesequences. “BLAST 2 Sequences” can be accessed and used interactively atthe NCBI website. The “BLAST 2 Sequences” tool can be used for bothblastn and blastp (discussed above).

Regarding polynucleotide sequences, percent identity may be measuredover the length of an entire defined polynucleotide sequence, forexample, as defined by a particular SEQ ID number, or may be measuredover a shorter length, for example, over the length of a fragment takenfrom a larger, defined sequence, for instance, a fragment of at least20, at least 30, at least 40, at least 50, at least 70, at least 100, orat least 200 contiguous nucleotides. Such lengths are exemplary only,and it is understood that any fragment length supported by the sequencesshown herein, in the tables, figures, or Sequence Listing, may be usedto describe a length over which percentage identity may be measured.

Regarding polynucleotide sequences, “variant,” “mutant,” or “derivative”may be defined as a nucleic acid sequence having at least 50% sequenceidentity to the particular nucleic acid sequence over a certain lengthof one of the nucleic acid sequences using blastn with the “BLAST 2Sequences” tool available at the National Center for BiotechnologyInformation's website. (See Tatiana A. Tatusova, Thomas L. Madden(1999), “Blast 2 sequences—a new tool for comparing protein andnucleotide sequences”, FEMS Microbiol Lett. 174:247-250). Such a pair ofnucleic acids may show, for example, at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% or greater sequence identity over a certaindefined length.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof the genetic code where multiple codons may encode for a single aminoacid. It is understood that changes in a nucleic acid sequence can bemade using this degeneracy to produce multiple nucleic acid sequencesthat all encode substantially the same protein. For example,polynucleotide sequences as contemplated herein may encode a protein andmay be codon-optimized for expression in a particular host. In the art,codon usage frequency tables have been prepared for a number of hostorganisms including humans, mouse, rat, pig, E. coli, plants, and otherhost cells.

A “recombinant nucleic acid” is a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques known in the art. Theterm recombinant includes nucleic acids that have been altered solely byaddition, substitution, or deletion of a portion of the nucleic acid.Frequently, a recombinant nucleic acid may include a nucleic acidsequence operably linked to a promoter sequence. Such a recombinantnucleic acid may be part of a vector that is used, for example, totransform a cell.

The nucleic acids disclosed herein may be “substantially isolated orpurified.” The term “substantially isolated or purified” refers to anucleic acid that is removed from its natural environment, and is atleast 60% free, preferably at least 75% free, and more preferably atleast 90% free, even more preferably at least 95% free from othercomponents with which it is naturally associated.

“Transformation” or “transfected” describes a process by which exogenousnucleic acid (e.g., DNA or RNA) is introduced into a recipient cell.Transformation or transfection may occur under natural or artificialconditions according to various methods well known in the art, and mayrely on any known method for the insertion of foreign nucleic acidsequences into a prokaryotic or eukaryotic host cell. The method fortransformation or transfection is selected based on the type of hostcell being transformed and may include, but is not limited to,bacteriophage or viral infection or non-viral delivery. Methods ofnon-viral delivery of nucleic acids include lipofection, nucleofection,microinjection, electroporation, heat shock, particle bombardment,biolistics, virosomes, liposomes, immunoliposomes, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection is described in e.g., U.S.Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagentsare sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91/17424; WO91/16024. Delivery can be to cells (e.g. in vitro or ex vivoadministration) or target tissues (e.g. in vivo administration). Theterm “transformed cells” or “transfected cells” includes stablytransformed or transfected cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome, as well as transiently transformed or transfectedcells which express the inserted DNA or RNA for limited periods of time.

The polynucleotide sequences contemplated herein may be present inexpression vectors. For example, the vectors may comprise: (a) apolynucleotide encoding an ORF of a protein; (b) a polynucleotide thatexpresses an RNA that directs RNA-mediated binding, nicking, and/orcleaving of a target DNA sequence; and both (a) and (b). Thepolynucleotide present in the vector may be operably linked to aprokaryotic or eukaryotic promoter. “Operably linked” refers to thesituation in which a first nucleic acid sequence is placed in afunctional relationship with a second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences may be in close proximity or contiguousand, where necessary to join two protein coding regions, in the samereading frame. Vectors contemplated herein may comprise a heterologouspromoter (e.g., a eukaryotic or prokaryotic promoter) operably linked toa polynucleotide that encodes a protein. A “heterologous promoter”refers to a promoter that is not the native or endogenous promoter forthe protein or RNA that is being expressed. For example, a heterologouspromoter for a LAMP may include a eukaryotic promoter or a prokaryoticpromoter that is not the native, endogenous promoter for the LAMP.

As used herein, “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into and mRNAor other RNA transcript) and/or the process by which a transcribed mRNAis subsequently translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA,expression may include splicing of the mRNA in a eukaryotic cell.

The term “vector” refers to some means by which nucleic acid (e.g., DNA)can be introduced into a host organism or host tissue. There are varioustypes of vectors including plasmid vector, bacteriophage vectors, cosmidvectors, bacterial vectors, and viral vectors. As used herein, a“vector” may refers to a recombinant nucleic acid that has beenengineered to express a heterologous polypeptide (e.g., the fusionproteins disclosed herein). The recombinant nucleic acid typicallyincludes cis-acting elements for expression of the heterologouspolypeptide.

Any of the conventional vectors used for expression in eukaryotic cellsmay be used for directly introducing DNA into a subject. Expressionvectors containing regulatory elements from eukaryotic viruses may beused in eukaryotic expression vectors (e.g., vectors containing SV40,CMV, or retroviral promoters or enhancers). Exemplary vectors includethose that express proteins under the direction of such promoters as theSV40 early promoter, SV40 later promoter, metallothionein promoter,human cytomegalovirus promoter, murine mammary tumor virus promoter, andRous sarcoma virus promoter. Expression vectors as contemplated hereinmay include eukaryotic or prokaryotic control sequences that modulateexpression of a heterologous protein (e.g. the fusion protein disclosedherein). Prokaryotic expression control sequences may includeconstitutive or inducible promoters (e.g., T3, T7, Lac, trp, or phoA),ribosome binding sites, or transcription terminators.

The vectors contemplated herein may be introduced and propagated in aprokaryote, which may be used to amplify copies of a vector to beintroduced into a eukaryotic cell or as an intermediate vector in theproduction of a vector to be introduced into a eukaryotic cell (e.g.amplifying a plasmid as part of a viral vector packaging system). Aprokaryote may be used to amplify copies of a vector and express one ormore nucleic acids, such as to provide a source of one or more proteinsfor delivery to a host cell or host organism. Expression of proteins inprokaryotes may be performed using Escherichia coli with vectorscontaining constitutive or inducible promoters directing the expressionof either a protein or a fusion protein comprising a protein or afragment thereof. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; (iii) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification (e.g., a His tag); (iv) to tag the recombinantprotein for identification (e.g., such as Green fluorescence protein(GFP) or an antigen (e.g., HA) that can be recognized by a labelledantibody); (v) to promote localization of the recombinant protein to aspecific area of the cell (e.g., where the protein is fused (e.g., atits N-terminus or C-terminus) to a nuclear localization signal (NLS)which may include the NLS of SV40, nucleoplasmin, C-myc, M9 domain ofhnRNP A1, or a synthetic NLS). The importance of neutral and acidicamino acids in NLS have been studied. (See Makkerh et al. (1996) CurrBiol 6(8):1025-1027). Often, in fusion expression vectors, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase.

The presently disclosed methods may include delivering one or morepolynucleotides, such as or one or more vectors as described herein, oneor more transcripts thereof, and/or one or proteins transcribedtherefrom, to a host cell. Further contemplated are host cells producedby such methods, and organisms (such as animals, plants, or fungi)comprising or produced from such cells. The disclosed extracellularvesicles may be prepared by introducing vectors that express mRNAencoding a fusion protein and a cargo RNA as disclosed herein.Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids in mammalian cells or target tissues.Non-viral vector delivery systems include DNA plasmids, RNA (e.g. atranscript of a vector described herein), naked nucleic acid, andnucleic acid complexed with a delivery vehicle, such as a liposome.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell.

In the methods contemplated herein, a host cell may be transiently ornon-transiently transfected (i.e., stably transfected) with one or morevectors described herein. In some embodiments, a cell is transfected asit naturally occurs in a subject (i.e., in situ). In some embodiments, acell that is transfected is taken from a subject (i.e., explanted). Insome embodiments, the cell is derived from cells taken from a subject,such as a cell line. Suitable cells may include stem cells (e.g.,embryonic stem cells and pluripotent stem cells). A cell transfectedwith one or more vectors described herein may be used to establish a newcell line comprising one or more vector-derived sequences. In themethods contemplated herein, a cell may be transiently transfected withthe components of a system as described herein (such as by transienttransfection of one or more vectors, or transfection with RNA), andmodified through the activity of a complex, in order to establish a newcell line comprising cells containing the modification but lacking anyother exogenous sequence.

EXAMPLES

The following Examples are illustrative and are not intended to limitthe scope of the claimed subject matter.

Example 1—A Targeted and Modular Exosome Loading (TAMEL) System

Reference is made to U.S. Published Patent Application No. 2015/0093433,published on Apr. 2, 2015, and Hung and Leonard, “A platform foractively loading cargo RNA to elucidate limiting steps in EV-mediateddelivery,” J. Extracellular Vesicles, 2016, 5: 31027, published 13 May2016, the contents of which are incorporated herein by reference intheir entireties.

Abstract

This Example relates to a Targeted and Modular Exosome Loading (TAMEL)system, which is a technology for directing the loading of RNA intoexosomes. Secreted extracellular vesicles are emerging as important newfeatures of the expanding landscape of intercellular communication. Theprocess of secretion of exosomes by an exosome-producing cell and theprocess of uptake of the secreted exosomes by a recipient cell areillustrated schematically in FIGS. 1 and 2. A subset of extracellularvesicles in the 30-200 nanometer diameter range, known as exosomes, havebeen found to play a number of important roles in intercellularsignaling, including shedding of obsolete proteins during reticulocytematuration [1], presentation of antigens to T cells [2], activation of Band T cell proliferation [3], and induction of immune rejection ofmurine tumors, presumably by delivery or presentation of tumor antigensto the immune system [4]. Exosomes have generated great interest fortheir roles in intercellular communication and their potential totherapeutically modulate immune cell signaling. Subsequentinvestigations into exosome biogenesis, cargo packaging, and mediationof intercellular communication have identified new opportunities forharnessing and modifying exosomes to develop exosome-based therapeutics.

The TAMEL system disclosed here utilizes a “packaging protein” and a“cargo RNA.” The “packaging protein” may be referred to as a EV-loadingprotein. (See Hung and Leonard, “A platform for actively loading cargoRNA to elucidate limiting steps in EV-mediated delivery,” J.Extracellular Vesicles, 2016, 5: 31027, published 13 May 2016, thecontent of which is incorporated herein by reference in its entirety.)The packaging protein is an RNA-binding protein targeted to exosomes viafusion to an exosome-targeted domain of a lysosomal protein. The cargoRNA is an RNA molecule displaying the proper RNA-motif for binding bythe packaging protein. This packaging system is novel in that it is thefirst method by which any type of RNA (e.g., miRNA, shRNA, mRNA, ncRNA)can be targeted for loading into exosomes via fusion to the RNA-motif,without the need for overexpression of the RNA of interest.Overexpression generally is disfavored because it can alter thephysiology of the exosome-producing cell. The ability to selectivelyenrich RNAs in exosomes is essential to the engineering of exosomes astherapeutic delivery vehicles. RNA-loaded exosomes have a wide varietyof potential therapeutic uses and are already being investigated asdelivery vehicles for gene therapy, vaccines, and reprogramming factorsin the generation of pluripotent stem cells. However, the therapeuticutility of exosomes is hampered by a general lack of control over whichmolecules are loaded from the parent cell into the exosomes. Thetechnology disclosed herein provides the capability to control which RNAspecies are most abundant in exosomes.

DESCRIPTION

In this example, the TAMEL packaging protein consists of an RNA-bindingprotein fused to Lamp2b. Lamp2b has been previously shown to localize toexosomes [5]. Alvarez-Erviti et al. determined the orientation of Lamp2bin exosomes (N-terminus on the exterior of exosomes, C-terminus on theinterior of exosomes) and showed that peptides fused to the N-terminusof Lamp2b could be displayed on the outside of exosomes [5]. (See FIG. 3for schematic examples of Lamp2b fusion proteins for expressing aprotein of interest on the surface of an exosome versus the lumen of theexosome). To direct the loading of RNA into the lumen of the exosome, wefused an RNA-binding protein to the C-terminus of Lamp2b. (See FIG. 4).We have tested the system using RNA-binding proteins that have beenpreviously characterized, including bacteriophage coat proteins from theMS2 and LambaN bacteriophages [6].

The TAMEL system of this Example may be implemented as follows: (a) anRNA-binding protein, such as a bacteriophage coat protein, is chosen;(b) a packaging protein comprising Lamp2b on the N-terminus and theRNA-binding protein on the C-terminus is designed (see FIG. 4); (c)cargo RNA containing the packaging protein binding motif is designed(see FIG. 4); (d) DNA sequences encoding the packaging protein and cargoRNA are generated (by molecular biology and/or DNA synthesis) andinserted into a suitable expression vector (e.g., viral vector for cargoRNA, plasmid or viral vector for packaging protein); (e) the cargo RNAexpression vector is transduced into a suitable cell for producing RNA(or RNA is produced in vitro and transduced into a suitable cell forproducing exosomes) and the packaging protein vector is transfected ortransduced into a suitable cell line for producing exosomes; (f)exosomes are harvesting from the cell line producing the exosomes; and(g) RNA is isolated from these exosomes and quantified by qPCR.

The mode of action of the TAMEL system is that the packaging proteinfused to Lamp2b is capable both of localizing to exosomes through itsLamp2b domain and binding RNA through its RNA-binding domain. During theprocess of exosome biogenesis, the RNA-binding domain is initiallylocalized in the cytoplasm, where it has access to cytoplasmic RNAspecies, including the cargo RNA. The inward budding of themultivesicular body (MVB) membrane to form intraluminal vesicles (ILVs),results in the RNA-binding domain localizing in the lumen of ILVs. BoundRNA should move in concert with the RNA-binding domain, also localizingto the ILV lumen. As ILVs are released from the exosome-producing cell,as exosomes, the RNA-binding domain and bound RNA remain in the vesiclelumen, ultimately resulting in their presence in the lumen of exosomes.(See FIG. 1). After being released from an exosome-producing cell, theexosomes may be delivered to a target cell (i.e., recipient cell) wherethe exosomes are taken up and the exosome cargo is delivered to thecytoplasm of the target cell. (See FIG. 2).

As illustrated in FIG. 5, cargo RNA bearing the MS2 RNA-binding loop wastransduced into cells at high or low copy number, either in the presenceor absence of the TAMEL packaging protein bearing the RNA-binding domainof the coat protein of MS2. The cargo RNA was a 187 base pair small RNAdisplaying the high affinity MS2 binding loop (HA MS2 Loop). The cargoRNA was transduced into cells at high copy or low copy number for highor low expression, respectively. Cargo RNA levels were normalized toGAPDH reference RNA in (top) cells and (bottom) exosomes. An observedincrease in cargo RNA level in exosomes was significant by a student'st-test at a p-value of 0.05. Therefore, the TAMEL system increased theincorporation of a small (˜190 bp) RNA into exosomes. In the presence ofthe Lamp2b-MS2 TAMEL packaging protein, the small cargo RNA levelincreased 1.4-4.4 fold in exosomes compared to no TAMEL packagingprotein.

To investigate whether “longer” cargo RNA could be incorporated intoexosomes using the TAMEL system, we engineered lentiviral vectorsdriving expression of cargo RNA (˜1700 nt plus 100-250 poly-A) via RNAPol II. These cargo RNAs had either no MS2 binding loop or a highaffinity MS2 binding loop, facilitating the cargo RNA to be bound by theLamp2b-MS2 TAMEL packaging protein. We transfected cell lines with theLamp2b-MS2 TAMEL packaging protein or a negative control protein(Lamp2b-neg). (See FIG. 4). Despite the fact that the cells transfectedwith the TAMEL packaging protein had lower levels of cargo RNA thanthose transfected with the negative control protein (FIG. 4, top), thepresence of the TAMEL packaging protein increased the incorporation ofthe cargo RNA into exosomes by about 7 fold (FIG. 4, bottom versus top).These results indicate that the TAMEL system can be applied to packagelarge RNAs into exosomes.

DISCUSSION

The TAMEL system disclosed here offers advantages over two existingmethods for enriching RNAs in exosomes: (1) overexpression and (2) RNAzipcodes [7]. Overexpression is a commonly utilized strategy forincorporating RNA into exosomes which comprises simply overexpressingthe cargo RNA in the exosome-producing cells. This method potentiallyutilizes a mass action driving force to promote nonspecificincorporation of cargo RNA into exosomes. Such cargo RNA overexpressionin producer cells has been used to incorporate miRNA [8], [9], [10],chemically modified 3′ benzen-pyridine miRNA [11], shRNA [9], and mRNA[12],[13] into exosomes. Upon incubation of exosomes carrying these RNAswith recipient cells, these overexpressed RNAs were all functional (i.e.the mRNA was translated into protein, and the shRNAs and miRNAs inducedtarget gene knockdown). This strategy thus appears to be broadlyapplicable to a variety of RNA cargos and recipient cell types.

Nonetheless, this technique has not been explored broadly enough todetermine whether it is robust and widely applicable. The observationthat some RNA species that are highly abundant in cells are not presentin the exosomes produced from those cells [14], [15], [16], [17]suggests that this strategy may have varying degrees of success fordifferent types of RNA, and indeed may be incapable of mediating thepackaging of RNAs that may be actively excluded from exosomes.Furthermore, overexpression of RNA can impact host cell physiology,causing changes in cell health, or possibly changes in exosomeproduction itself. These effects may hinder the packaging of certainRNAs into exosomes, for example therapeutic RNAs intended to induceapoptosis in cancer cells. In contrast, the described TAMEL system isnot dependent on high expression levels of the cargo RNA. In fact, theplatform could be engineered for greater sensitivity to RNAs that areexpressed only at low levels (for example, by engineering higheraffinity RNA-binding domains). Furthermore, because TAMEL is independentof host packaging mechanisms, it is capable of loading any RNA intoexosomes, even RNAs that have been observed to be excluded fromexosomes.

RNA zipcodes refer to structural and sequence motifs that have beenidentified as enriched in exosomes, and may be utilized to direct theloading of RNA into exosomes. For example, deep sequencing of exosomeRNA revealed that miRNAs with 3′ modifications are enriched inexosomes[18]. Potentially, 3′ modification of miRNA could be used toload specific miRNAs into exosomes, but this has not been tested. In thecase of mRNA, however, RNA zipcodes have been used to enrich mRNA inexosomes. RNA zipcodes are sequence motifs in the 3′ untranslated region(UTR) that direct mRNA localization within the cell. Bolukbasi et al.identified two features—a miR-1289 binding site and a core “CTGCC”motif—that are enriched in the 3′ UTRs of a large proportion of mRNAsfound in glioblastoma- and melanoma-derived exosomes. Replacing the 3′UTR of eGFP with a 25 nucleotide sequence containing the miR-1289binding site and the “CTGCC” motif added was sufficient to increase eGFPmRNA incorporation into HEK293T exosomes by 2-fold compared to untaggedeGFP mRNA. Overexpression of miR-1289 further increased theincorporation of the construct 6-fold compared to the untagged eGFPmRNA. This increase in exosome targeting depended on the presence of themiR-1289 binding site, as mutation of this site abrogated enrichment ofthe mRNA in exosomes [7]. This approach to RNA loading applies only tomRNA, which contain a 3′ UTR. Whether or not these zipcodes could beplaced in non-coding and small RNAs to mediate loading into exosomes isunknown. Furthermore, overexpression of miR-1289 increases the levels ofendogenous mRNAs containing miR-1289 binding sites loaded into exosomes[7] which could be undesirable for certain applications.

In contrast to these RNA-motifs, the TAMEL system can be applied to anytype of RNA and does require interfering with native exosome loadingmechanisms. As such, the TAMEL system is widely applicable. For example,the TAMEL system may be used: (a) to enrich exosomes with therapeuticRNA for use of exosomes as gene therapy delivery vehicles; (b) to enrichexosomes with RNA as part of an exosome vaccine; (c) to enrich exosomeswith reprogramming RNAs for generating pluripotent stem cells; (d) toenrich exosomes with a specific RNA for delivering the RNA to recipientcells as an alternative to transfection or transduction; (e) to studyand characterize the factors that affect loading of native RNA intoexosomes via using the TAMEL system as a model and modifying variousaspects of the TAMEL system to determine how the modifications affectRNA loading.

Notably, as demonstrated here, the present TAMEL system can be utilizedto incorporate relatively long mRNAs. This result is important becausethis indicates that the TAMEL system will be useful for designingexosomes for delivering mRNAs to target cells, which could be useful ina variety of therapeutic applications.

REFERENCES

-   1. Johnstone, R. M., et al., Vesicle formation during reticulocyte    maturation. Association of plasma membrane activities with released    vesicles (exosomes). J Biol Chem, 1987. 262(19): p. 9412-20.-   2. Raposo, G., et al., B lymphocytes secrete antigen-presenting    vesicles. J Exp Med, 1996. 183(3): p. 1161-72.-   3. Skokos, D., et al., Mast cell-dependent B and T lymphocyte    activation is mediated by the secretion of immunologically active    exosomes. J Immunol, 2001. 166(2): p. 868-76.-   4. Zitvogel, L., et al., Eradication of established murine tumors    using a novel cell-free vaccine: dendritic cell-derived exosomes.    Nat Med, 1998. 4(5): p. 594-600.-   5. Alvarez-Erviti, L., et al., Delivery of siRNA to the mouse brain    by systemic injection of targeted exosomes. Nat Biotechnol, 2011.    29(4): p. 341-5.-   6. Keryer-Bibens, C., C. Barreau, and H. B. Osborne, Tethering of    proteins to RNAs by bacteriophage proteins. Biol Cell, 2008.    100(2): p. 125-38.-   7. Bolukbasi, M. F., et al., miR-1289 and “Zipcode”-like Sequence    Enrich mRNAs in Microvesicles. Mol Ther Nucleic Acids, 2012. 1: p.    e10.-   8. Ohno, S., et al., Systemically injected exosomes targeted to EGFR    deliver antitumor microRNA to breast cancer cells. Mol Ther, 2013.    21(1): p. 185-91.-   9. Rechavi, O., et al., Cell contact-dependent acquisition of    cellular and viral nonautonomously encoded small RNAs. Genes    Dev, 2009. 23(16): p. 1971-9.-   10. Kosaka, N., et al., Competitive interactions of cancer cells and    normal cells via secretory microRNAs. J Biol Chem, 2012. 287(2): p.    1397-405.-   11. Akao, Y., et al., Microvesicle-mediated RNA molecule delivery    system using monocytes/macrophages. Mol Ther, 2011. 19(2): p. 395-9.-   12. Hergenreider, E., et al., Atheroprotective communication between    endothelial cells and smooth muscle cells through miRNAs. Nat Cell    Biol, 2012. 14(3): p. 249-56.-   13. Mizrak, A., et al., Genetically engineered microvesicles    carrying suicide mRNA/protein inhibit schwannoma tumor growth. Mol    Ther, 2013. 21(1): p. 101-8.-   14. Valadi, H., et al., Exosome-mediated transfer of mRNAs and    microRNAs is a novel mechanism of genetic exchange between cells.    Nat Cell Biol, 2007. 9(6): p. 654-9.-   15. Montecalvo, A., et al., Mechanism of transfer of functional    microRNAs between mouse dendritic cells via exosomes. Blood, 2012.    119(3): p. 756-66.-   16. Iguchi, H., N. Kosaka, and T. Ochiya, Secretory microRNAs as a    versatile communication tool. Commun Integr Biol, 2010. 3(5): p.    478-81.-   17. Kucharzewska, P., et al., Exosomes reflect the hypoxic status of    glioma cells and mediate hypoxia-dependent activation of vascular    cells during tumor development. Proc Natl Acad Sci USA, 2013.    110(18): p. 7312-7.-   18. Koppers-Lallic, D. H., M.; van Eijndhoven, M. E.; Sabogal    Pineros, Y.; Sie, D.; Ylstra, B.; Middeldorp, J. M.; Pegtel, D. M.,    Comprehensive deep-sequencing analysis reveals non-random small RNA    incorporation into tumour exosomes and biomarker potential. Journal    of Extracellular Vesicles, 2013. 2: p. 20826.-   19. Lotvall, J. O. V., H., Exosome transfer of nucleic acids to    cells, USPTO, 2007.

Example 2—A CD63-MS2 Fusion Protein as an RNA Packaging Protein forExosomes

We previously demonstrated that the exosome-targeting domain of theprotein Lamp2b can be used as a packaging protein for exosomes. (SeeU.S. Published Application No. 20150093433, the content of which isincorporated herein by reference in its entirety). We showed that Lamp2bfused to MS2 significantly enhances cargo RNA loading into exosomes. Inthis Example, we found that CD63 protein also includes a potentialexosome-targeting domain. CD63 fused to MS2 significantly enhances cargoRNA loading into exosomes.

The MS2 coat protein dimer used previously was genetically fused to theC-terminus of CD63, including a 7 amino acid flexible spacer between theCD63 C terminus and the MS2 dimer N terminus, and an human influenzahemagglutanin (HA) affinity tag was added to the C-terminus of MS2. TheHA affinity tag does not affect the function of MS2 and was included toenable using Western blot experiments to verify the presence of thisengineered protein in exosomes. A similar control construct wasgenerated by fusing the HA affinity tag (alone) to CD63 in place of“MS2-HA”.

These proteins were expressed by transient transfection in the existingHEK293FT-based cell line, which constitutively expresses adTomato-encoding cargo RNA that includes 3 MS2 binding loops in the 3′untranslated region. We then harvested exosomes from cells expressingthe cargo RNA along with either CD63-MS2-HA or CD63-HA, and we used qPCRto determine the ratio of dTomato cargo RNA to GAPDH mRNA in theresulting exosome samples. This analysis revealed increased loading ofthe cargo RNA (per GAPDH mRNA) into exosomes when the exosome-producingcells expressed the CD63-MS2-HA protein rather than the CD63-HA controlprotein. (See FIG. 7).

Example 3—A VSVG-MS2 Fusion Protein as an RNA Packaging Protein forExosomes

Cells transfected with Vesicular Stomatis Virus G protein (VSVG) produceextracellular vesicles called “gesicles.” In this Example, we found thata VSVG-MS2 fusion significantly enhances cargo RNA loading intogesicles.

MS2-HA or HA was genetically fused to the C-terminus of VSVG in the samemanner as was used to engineer CD63, as described above. Similarexperiments were used to evaluated loading, except that the vesicleswere harvested according to the protocol previously developed forharvesting vesicles containing VSVG (“gesicles”) (See Mangeot et al.,Molecular Therapy (2011) 19:9, 1656-1666).

Briefly, the difference between this protocol and the standard exosomepurification protocol involved the use of centrifugation steps ofdifferent speeds and durations, such that potentially, a different poolof vesicles may be obtained. As observed for the CD63 experiments,expression of the VSVG-MS2-HA construct led to enhanced loading of thecargo RNA into vesicles (relative to cells expressing the VSVG-HAcontrol protein). (See FIG. 8).

REFERENCES

-   1. Johnstone, R. M., et al., Vesicle formation during reticulocyte    maturation. Association of plasma membrane activities with released    vesicles (exosomes). J Biol Chem, 1987. 262(19): p. 9412-20.-   2. Raposo, G., et al., B lymphocytes secrete antigen-presenting    vesicles. J Exp Med, 1996. 183(3): p. 1161-72.-   3. Skokos, D., et al., Mast cell-dependent B and T lymphocyte    activation is mediated by the secretion of immunologically active    exosomes. J Immunol, 2001. 166(2): p. 868-76.-   4. Zitvogel, L., et al., Eradication of established murine tumors    using a novel cell-free vaccine: dendritic cell-derived exosomes.    Nat Med, 1998. 4(5): p. 594-600.-   5. Alvarez-Erviti, L., et al., Delivery of siRNA to the mouse brain    by systemic injection of targeted exosomes. Nat Biotechnol, 2011.    29(4): p. 341-5.-   6. Keryer-Bibens, C., C. Barreau, and H. B. Osborne, Tethering of    proteins to RNAs by bacteriophage proteins. Biol Cell, 2008.    100(2): p. 125-38.-   7. Bolukbasi, M. F., et al., miR-1289 and “Zipcode”-like Sequence    Enrich mRNAs in Microvesicles. Mol Ther Nucleic Acids, 2012. 1: p.    e10.-   8. Ohno, S., et al., Systemically injected exosomes targeted to EGFR    deliver antitumor microRNA to breast cancer cells. Mol Ther, 2013.    21(1): p. 185-91.-   9. Rechavi, O., et al., Cell contact-dependent acquisition of    cellular and viral nonautonomously encoded small RNAs. Genes    Dev, 2009. 23(16): p. 1971-9.-   10. Kosaka, N., et al., Competitive interactions of cancer cells and    normal cells via secretory microRNAs. J Biol Chem, 2012. 287(2): p.    1397-405.-   11. Akao, Y., et al., Microvesicle-mediated RNA molecule delivery    system using monocytes/macrophages. Mol Ther, 2011. 19(2): p. 395-9.-   12. Hergenreider, E., et al., Atheroprotective communication between    endothelial cells and smooth muscle cells through miRNAs. Nat Cell    Biol, 2012. 14(3): p. 249-56.-   13. Mizrak, A., et al., Genetically engineered microvesicles    carrying suicide mRNA/protein inhibit schwannoma tumor growth. Mol    Ther, 2013. 21(1): p. 101-8.-   14. Valadi, H., et al., Exosome-mediated transfer of mRNAs and    microRNAs is a novel mechanism of genetic exchange between cells.    Nat Cell Biol, 2007. 9(6): p. 654-9.-   15. Montecalvo, A., et al., Mechanism of transfer of functional    microRNAs between mouse dendritic cells via exosomes. Blood, 2012.    119(3): p. 756-66.-   16. Iguchi, H., N. Kosaka, and T. Ochiya, Secretory microRNAs as a    versatile communication tool. Commun Integr Biol, 2010. 3(5): p.    478-81.-   17. Kucharzewska, P., et al., Exosomes reflect the hypoxic status of    glioma cells and mediate hypoxia-dependent activation of vascular    cells during tumor development. Proc Natl Acad Sci USA, 2013.    110(18): p. 7312-7.-   18. Koppers-Lallic, D. H., M.; van Eijndhoven, M. E.; Sabogal    Pineros, Y.; Sie, D.; Ylstra, B.; Middeldorp, J. M.; Pegtel, D. M.,    Comprehensive deep-sequencing analysis reveals non-random small RNA    incorporation into tumour exosomes and biomarker potential. Journal    of Extracellular Vesicles, 2013. 2: p. 20826.-   19. Lotvall, J. O. V., H., Exosome transfer of nucleic acids to    cells, U.S. Published Patent Application No. 2007/0298118, 2007.-   20. Hung, M. E. and Leonard, J., Stabilization of Exosome-targeting    Peptides via Engineered Glycosylation, J. Biol. Chem., Vol. 290, NO.    13, pp. 8166-8172, Mar. 27, 2015.-   21. Schulz, Chapter 2: Beyond the Sequon: Sites of N-Glycosylation,    Biochemistry, Genetics and Molecular Biology “Glycosylation,” edited    by Stefana Petrescu, Sep. 26, 2012.-   22. Bano-Polo, M., et al., N-Glycosylation efficiency is determined    by the distance to the C-terminus and the amino acid preceding an    Asn-Ser-Thr sequon, Protein Sci. 20, 179-186 (2011).-   23. Kundra, R., et al., Asparagine-linked oligosaccharides protect    Lamp-1 and Lamp-2 from intracellular proteolysis, J. Biol. Chem.    274, 31039-31046 (1999).

Example 5—Stabilization of Exosome-Targeting Peptides Via EngineeredGlycosylation

Reference is made to Hung, M. E. and Leonard, J., Stabilization ofExosome-targeting Peptides via Engineered Glycosylation, J. Biol. Chem.,Vol. 290, NO. 13, pp. 8166-8172, Mar. 27, 2015, the content of which isincorporated herein by reference in its entirety.

Abstract

Exosomes are secreted extracellular vesicles that mediate intercellulartransfer of cellular contents and are attractive vehicles fortherapeutic delivery of bimolecular cargo such as nucleic acids,proteins, and even drugs. Efficient exosome-mediated delivery in vivorequires targeting vesicles for uptake by specific recipient cells.Although exosomes have been successfully targeted to several cellularreceptors by displaying peptides on the surface of the exosomes,identifying effective exosome-targeting peptides for other receptors hasproven challenging. Furthermore, the biophysical rules governingtargeting peptide success remain poorly understood. To evaluate onefactor potentially limiting exosome delivery, we investigated whetherpeptides displayed on the exosome surface are degraded during exosomebiogenesis, for example by endosomal proteases. Indeed, peptides fusedto the N terminus of exosome-associated transmembrane protein Lamp2bwere cleaved in samples derived from both cells and exosomes. Tosuppress peptide loss, we engineered targeting peptide-Lamp2b fusionproteins to include a glycosylation motif at various positions.Introduction of this glycosylation motif both protected the peptide fromdegradation and led to an increase in overall Lamp2b fusion proteinexpression in both cells and exosomes. Moreover,glycosylation-stabilized peptides enhanced targeted delivery of exosomesto neuroblastoma cells, demonstrating that such glycosylation does notablate peptide-target interactions. Thus, we have identified a strategyfor achieving robust display of targeting peptides on the surface ofexosomes, which should facilitate the evaluation and development of newexosome-based therapeutics.

INTRODUCTION

Lipid nanoparticles display many properties that make them excellentdrug delivery vehicles, including the ability to enhance drug stabilityand solubility and alter drug pharmacokinetics to achieve higher drugconcentrations in target tissues (1). Biologically derived nanoparticlesare an emerging subset of lipid nanoparticles that have been shown toeffectively deliver a wide range of functional biomolecules, evade ordampen immune responses, and accumulate in tumors (2-4). In particular,exosomes, which are endosomally derived secreted vesicles, have showngreat promise as therapeutic delivery vehicles (5, 6). Exosomes havebeen used to deliver therapeutic RNA to neurons (7), ovarian cancers(8), glioblastomas (9), and colon cancers (10); to deliver proteins toglioblastomas (9); and to deliver small molecule drugs to breast cancers(11) and glioblastomas (12). Based on these successes, exosomes are nowbeing investigated in clinical trials as delivery vehicles for cancervaccines and small molecule drugs (13).

An attractive property of lipid nanoparticle-based drug delivery is thepotential to target lipid nanoparticles for uptake by specific recipientcells by functionalizing these particles with ligands that bindreceptors on recipient cells. The addition of targeting ligands to lipidnanoparticles enhances their uptake and retention in the desiredrecipient cell type or tissue. The addition of peptide-based targetingligands to synthetic lipid nanoparticles is nontrivial as peptideligands affect the stability and material properties of the lipidnanoparticle and increase the complexity of synthesis (14). In contrast,displaying targeting ligands on exosomes is relatively simple becausepeptide ligands can be genetically fused to the extra-exosomal terminiof exosomal membrane proteins. This strategy has been applied to targetexosome uptake by neurons by fusing a rabies viral glycoprotein (RVG)²peptide to the N terminus of lysosomal associated membrane protein 2b(Lamp2b) (7). Such a fusion resulted in RVG peptide being displayed onthe surface of exosomes, leading to exosome uptake via the nicotinicacetylcholine receptor. Similarly, an internalizing RGD peptide fused tothe N terminus of Lamp2b was used to target exosomes to breast cancercells via αvβ3 integrins (11). One alternative to Lamp2b, thetransmembrane domain of platelet-derived growth factor receptor, hasalso been used as a fusion partner to display peptides on the surface ofexosomes (8). However, it is not known whether such platelet-derivedgrowth factor receptor fusion proteins localize to endosomally derivedexosomes or rather to extracellular vesicles that bud from the plasmamembrane. Finally, fusion of peptides to the C1C2 domain of lactadherinhas been used to display peptides on the surface of exosomes forvaccines (15). However, lactadherin is a membrane-associated protein(16), not an integral membrane protein. Thus, peptides fused tolactadherin may be closely associated with the membrane, rather thanfreely accessible to interact with cell receptors.

Despite these successes, achieving efficient exosome targeting viasurface display of targeting peptides is nontrivial. AlthoughAlvarez-Erviti et al. (7) achieved neuronal targeting of exosomes viadisplay of the RVG peptide, they were unable to achieve muscle targetingvia display of a muscle-specific peptide similarly fused to Lamp2b. Thissuggests that different target-binding peptides may have differentutility as exosome-targeting peptides, possibly due to variations intarget binding affinity or level of peptide display on the exosomesurface, or a combination of these factors. In this study, wedemonstrate that some peptides fused to the N terminus of Lamp2b are notdisplayed effectively on the surface of exosomes. However, this displaycan be enhanced by introducing frequently glycosylated motifs atparticular locations within the engineered fusion protein. Wehypothesize that engineered glycosylation protects the targetingpeptides from degradation in the endosomal system during exosomebiogenesis and secretion. We also demonstrate that someglycosylation-protected peptides retain the ability to bind their targetproteins and that this peptide protection strategy can be applied totargeting peptides displayed on the surface of exosomes.

Experimental Procedures

Plasmid Construction. Human Lamp2b cDNA was purchased from OpenBiosystems and inserted into pcDNA3.1+ Hygro backbone. Peptide tags andglycine-serine amino acid spacers were added to the N and C termini ofLamp2b by PCR. The following tags were used: FLAG (DYKDDDDK), HA(YPYDVPDYA), and the glycosylation sequon (GNSTM) (17). Primer sequencesare available upon request.

Cell Culture and Transfection. HEK293FT cells (Life Technologies) andNeuro2A cells (gift from Richard Morimoto) were maintained at 37° C. in5% CO₂ in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin,and 4 mm 1-glutamine HEK293FT cells were plated at ˜60% confluency in10- or 15-cm dishes, and 1-1.5 μg of DNA/ml was transfected using theCaCl₂—HEPES-buffered saline method.

Exosome Production and Characterization. Exosome-free medium wasgenerated by pelleting FBS-derived exosomes from DMEM containing 20% FBS(see exosome pelleting protocol below) and combining the clearedsupernatant with serum-free DMEM to achieve a final concentration of 10%FBS. HEK293FT cells were transfected with Lamp2b expression plasmids,and medium was changed to exosome-free medium 12-14 h aftertransfection. Conditioned medium was collected 2 days after mediumchange, and exosomes were concentrated by differential centrifugation.Conditioned medium was spun at 300×g for 10 min, 2,000×g for 10 min, and10,000×g for 30 min to remove cells, cell debris, and apoptotic bodies.From this supernatant, exosomes were pelleted at 120,416×g for 135 minusing an SW41 Ti rotor in an L-80 Optima XP ultracentrifuge (BeckmanCoulter). Exosome pellets were washed in 10 ml of PBS and pelleted againvia ultracentrifugation. Exosome morphology was evaluated bytransmission electron microscopy using a 4% uranyl acetate negativestain. Exosome size distribution was profiled by NanoSight (Malvern)analysis.

Immunoblotting and Pulldown Assays. For Western blot analysis, cellextracts were prepared by lysis with radioimmunoprecipitation assaybuffer. Exosomes were not lysed. Cell lysates, exosomes, and pulldownswere heated in Laemmli buffer at 70° C. Equal quantities of protein, asmeasured by BCA assay (Pierce), were loaded in each lane of a 4-15%gradient polyacrylamide gel (Bio-Rad). After transfer to a PVDF membrane(Bio-Rad), membranes were blocked for 1 h in 1% milk at roomtemperature, and then blotted with anti-HA (Cell Signaling Technology,C29F4), anti-FLAG (Abcam, ab1162), or anti-β-actin (Cell SignalingTechnology, 8H10D10) antibodies. Primary antibodies were detected withhorseradish peroxidase-conjugated anti-mouse (Invitrogen) or anti-rabbit(Invitrogen and Abcam) immunoglobulin G secondary antibody. For FLAGpulldown experiments, cell lysates or exosomes were precleared withSepharose beads (Sigma) and then pulled down with FLAG M2 Sepharosebeads (Sigma) and eluted with 3×FLAG peptide. When indicated, celllysates were diluted 1:5 in TBS.

Inhibition of Endosomal Degradation. HEK293FT cells were transfectedwith Lamp2b expression plasmids. Cells were treated with 50 nmbafilomycin A1 (Sigma) or an equivalent amount of dimethyl sulfoxide(DMSO) for 9 h, or treated with 50 μm leupeptin (Sigma) for 24 h. Cellswere then lysed and evaluated by immunoblotting as described above.

Measuring Exosome Uptake. After the first ultracentrifuge spin of theexosome isolation procedure described above, exosome pellets (˜0.5 ml)were brought up to 1 ml in Diluent C (PKH67 kit, Sigma). This solutionwas mixed with 1 ml of Diluent C containing 6 μl of PKH67 dye and mixedvia constant pipetting for 1 min Next, 2 ml of 1% BSA was added to haltstaining. 4.5 ml of serum-free medium was added to bring the mixture upto 8.5 ml. Excess PKH67 dye can form micelles similar in size toexosomes, and these micelles cannot be separated from exosomes byultracentrifugation alone (18). We observed that these micelles are lessdense than exosomes (in 0.971 m sucrose, dye micelles float and exosomespellet).⁴ Thus, exosomes were purified via centrifugation through a0.971 m sucrose cushion. Briefly, 1.5 ml of 0.971 m sucrose was slowlypipetted underneath the 8.5 ml of exosome solution containing BSA andexcess dye. The entire mixture was centrifuged at 191,287×g for 2 h, andthen the upper layer and interface were carefully aspirated. To generatea negative control for estimating uptake of dye micelles, 0.5 ml ofserum-free medium was treated as an exosome pellet, labeled with PKH67,and subsequently processed exactly as were the exosome pellet samples.After purification through the sucrose cushion, exosomes were diluted1:10 in PBS and re-concentrated in 100-kDa cut-off centrifugal filterunits (Millipore UFC910024) to reduce the concentration of sucrose.Exosome concentration was then counted via NanoSight, and an equalnumber of exosomes from each sample (or all of the medium negativecontrol, to be conservative) were added to Neuro2A recipient cells.Recipient cells were plated at 50% confluency in a 48-well plate.Exosomes and cells were incubated for 2 h at 37° C., as described (11).Cells were then washed with PBS and harvested for flow cytometry. Flowcytometry was performed on an LSRII flow cytometer (BD Bioscience)running FACSDiva software. Data were analyzed using FlowJo software(TreeStar). Live single cells were gated based upon scatter.

Results

Peptides Fused to the N Terminus of Lamp2b Are Not Detected in Cells andExosomes. We hypothesized that peptides fused to the N terminus ofLamp2b would be vulnerable to proteolysis due to their localization inthe lumen of endosomes (FIG. 9A). Indeed, when glycosylation isartificially inhibited, Lamp2b is also vulnerable to degradation byendosomal proteases (19). To test this hypothesis, HEK293FT cells weretransfected with Lamp2b containing a C-terminal HA tag (Lamp2b-HA), twoC-terminal tags (Lamp2b-HA-FLAG), or an N-terminal FLAG tag and aC-terminal HA tag (FLAG-Lamp2b-HA). Cell lysates and exosomes wereharvested from these cells, and N- and C-terminal tags were analyzed byWestern blots. Exosomes harvested from HEK293FT cells exhibited expectedmorphology and size (FIGS. 9, B and C). As compared with samples derivedfrom whole cells, exosome samples were enriched in the exosomal proteinCD63 relative to β-actin (FIG. 9D), as expected. The Lamp2b fusionproteins were expressed at similar levels in cells, as indicated by theHA Western blot. However, the FLAG peptide on FLAG-Lamp2b-HA could notbe detected, although the C-terminal FLAG peptide of Lamp2b-HA-FLAG wasdetected (FIG. 9E). The same pattern was observed in exosomes (FIG. 9F).Together, these data indicate that the FLAG tag was lost from the Nterminus of the Lamp2b protein.

A Glycosylation Motif Protects Peptides on the N Terminus of Lamp2b andEnhances Expression of Lamp2b Fusion Proteins. Glycosylation of theLamp2b protein protects it from proteolytic degradation (19). Therefore,we hypothesized that engineered glycosylation could also protectpeptides fused to the N terminus of Lamp2b from proteolysis. Toinvestigate, the amino acid sequence GNSTM was fused to the N terminusof FLAG-Lamp2b-HA. The NST sequence is a standard N-linked glycosylationsequon, and the amino acids G and M flanking the sequon may increaseglycosylation frequency in mammals (11). To investigate how proximity tothe GNSTM tag impacts targeting peptide stability and availability tobind cellular receptors, fusion proteins were engineered to includeflexible amino acid spacers of various lengths between the GNSTM motif,the FLAG tag, and Lamp2b. Fusion protein expression was assessed incells and exosomes. The GNSTM-tagged Lamp2b proteins accumulated to agreater level in cells than did Lamp2b-HA or FLAG-Lamp2b-HA (FIG. 10A),and this difference was not an artifact of differential transfectionefficiency (FIG. 10B). The GNSTM-tagged Lamp2b proteins could also bedetected by anti-FLAG Western blot in both cell lysates (FIG. 10C) andin exosomes (FIG. 10D). Notably, increased detection of the FLAG tag wasnot simply a consequence of increased protein expression (compareFLAG-Lamp2b-HA and GNSTM-3gs-FLAG-3gs-Lamp2b-HA in FIG. 10C, where 3gsindicates a flexible linker 3 amino acids in length comprising glycineand serine residues), although increased detection of the FLAG tag didcorrelate with increased protein expression. These results suggest thatthe GNSTM motif effectively confers protection of the N-terminal FLAGpeptide. To test whether endosomal acidification was required for lossof the N-terminal FLAG peptide, cells were treated with bafilomycin A1.Bafilomycin A1 treatment preserved the FLAG peptide on FLAG-Lamp2b-HAbut had no effect on either the FLAG peptide on the C terminus ofLamp2b-HA-FLAG or the glycosylated FLAG peptide. Inhibiting endosomalproteases with leupeptin yielded the same pattern (FIG. 10E). Together,these data confirm that peptides displayed on the N terminus of Lamp2bare degraded by acid-dependent proteolysis. Moreover, adding the GNSTMglycosylation motif protected such N-terminal peptides from thisdegradation.

Peptides Protected by a Glycosylation Tag Retain the Capacity to BindCognate Peptide-binding Proteins. We next investigated whether GNSTMtag-stabilized peptide-Lamp2b fusion proteins retained the capacity tointeract with binding partners. GNSTM-tagged FLAG-Lamp2b proteins weresuccessfully pulled down by anti-FLAG beads in both cell lysates andintact exosomes (FIGS. 11, A and B). To verify that pulldown wasFLAG-specific and not an artifact due to increased loading of the highlyexpressed GNSTM-tagged proteins, lysates from cells expressingGNSTM-tagged proteins were diluted 1:5 such that the amount of thisLamp2b fusion protein loaded into the pulldown was comparable with theamounts in FLAG-Lamp2b-HA and Lamp2b-HA samples. This pulldown indicatedthat the FLAG peptide was indeed necessary for pulldown (FIG. 11C).Moreover, FLAG-dependent pulldown efficiency was much greater for someGNSTM-tagged Lamp2b constructs as compared with the FLAG-Lamp2b-HAcontrol. Also, pulldown of intact exosomes required that FLAG be on theexosome exterior because exosomes from cells expressing Lamp2b-HA-FLAGwere not pulled down (FIG. 11D). Collectively, these results indicatethat the GNSTM motif can protect targeting peptides fused to the Nterminus of Lamp2b from degradation without precluding interactionsbetween these peptides and their cognate protein targets.

Targeting Peptides Protected by a Glycosylation Tag Enhance ExosomeUptake by Recipient Cells. We next evaluated whether our engineeredglycosylation approach is compatible with a demonstrated exosometargeting strategy. To this end, we utilized the system described byAlvarez-Erviti et al. (7), in which display of the RVG peptide onexosomes mediated delivery to Neuro2A neuroblastoma cells. The RVGpeptide was fused to the N terminus of Lamp2b along with either a GNSTMglycosylation motif or a mutated motif, GASTM, which is notglycosylated. Exosomes displaying GNSTM-FLAG-Lamp2b-HA were alsoincluded as negative controls. All exosomes were labeled with thelipophilic dye PKH67, and equal numbers of exosomes from each samplewere incubated with Neuro2A cells for 2 h. Notably, uptake of GNSTM-RVGexosomes exceeded uptake of either GASTM-RVG or GNSTM-FLAG exosomes(FIGS. 12, A and B). Moreover, only the glycosylated RVG peptidesmediated exosome uptake greater than was observed for negative controlexosomes. Thus, in this system, engineered glycosylation of thetargeting peptide did not prevent targeted exosome uptake, and indeedglycosylation was required to confer targeted exosome uptake.

DISCUSSION

In this study, we found that peptides expressed on the N terminus ofLamp2b are susceptible to acid-dependent proteolytic degradation andthat the glycosylation motif GNSTM protects such peptides fromdegradation. This strategy allows for the display of targeting peptideson the surface of exosomes. Furthermore, the GNSTM motif does notinterfere with interactions between targeting peptides and their cognateprotein targets. Indeed, in our hands, the GNSTM motif enhancedtargeting peptide-mediated exosome uptake. In our investigation, theGNSTM motif conferred protection to peptides over a distance of at least10 flexible amino acids, and larger spacers may also be feasible. Thus,there may exist a substantial design space for applying this strategy tomany peptides of interest to ensure stable expression while avoidinginterference with peptide-target interactions, although someoptimization is likely to be required. Engineered glycosylation couldalso be useful for investigating and refining problematic exosometargeting strategies because without such modifications, candidatepeptides may fail to enhance exosome uptake due to peptide loss ratherthan due to problems inherent to the targeting approach.

In addition to protecting N-terminal peptides from degradation, addingthe GNSTM motif increased the total amount of Lamp2b fusion proteinpresent in cells and exosomes. This increase could be due to decreaseddegradation because increasing the glycosylation of Lamp1 and Lamp2during differentiation of the HL-60 promyelocytic cell line intogranulocytes increases the half-life of these proteins (20). However, inthis case, the complexity of the oligosaccharides on the proteins wasincreased, not the number of glycosylation sites. Because Lamp2b isalready expected to include 16-20 N-linked glycosylation sites (19),whether adding one additional glycosylation site could increase thehalf-life of Lamp2b is unclear. Glycosylation also plays a role in theexpression and sorting of other exosomal proteins. For example, sortingof the protein EWI-2 into exosomes is dependent on the presence ofcomplex N-linked glycans on this protein (21). Mutation of even one ofthree N-linked glycosylation sites significantly decreased EWI-2expression in extracellular vesicles. Furthermore, mutation of all threesites decreased both cellular and vesicle-associated EWI-2 levels. Thus,it is possible that altering the glycosylation of Lamp2b could havesimilar effects on expression in cells and exosomes.

Previous studies in which targeting peptides were fused to the Nterminus of Lamp2b did not report targeting peptide degradation,although these characterizations focused on evaluating whether targetingpeptide remained, rather than evaluating whether or not partialtargeting peptide degradation occurred (7, 11). Notably, neither of thepeptides used in these studies (RVG or internalizing RGD) contains anyputative N-linked glycosylation sites that could protect the peptidesfrom degradation. However, in these studies, exosomes wereelectroporated to load functional cargo molecules, and subsequentinvestigations have shown that electroporation can cause aggregation ofexosomes as well as RNA (22, 23). Thus, it is possible that suchaggregates may display different properties than did the exosomes usedin our experiments. Furthermore, these prior studies utilized the mouseLamp2b protein and isolated exosomes from murine dendritic cells,whereas we engineered human Lamp2b and isolated exosomes from humancells (HEK293FT). Thus, differences in protein trafficking and exosomesecretion in different species and cell types may result in differentsusceptibilities of targeting peptides to degradation. Alternatively,any factors leading to increased stability of mouse Lamp2b-basedconstructs may lead to accumulation of higher levels of Lamp2b in cellsand exosomes, such that some pool of intact peptide-Lamp2b escapesproteolytic cleavage prior to exosome secretion. Thus, the strategyproposed here may be especially important for engineering human Lamp2band human cell-derived exosomes, and whether these benefits may extendto murine exosome engineering remains to be determined.

Because targeting exosomes to specific receptors is required foreffective delivery of therapeutic molecules in vivo (7, 11), ourstrategy for increasing the expression of targeting peptides on exosomesmay be particularly useful for translating promising exosome-basedtherapeutic strategies from preclinical investigations to human trials.Even if mouse Lamp2b could confer enhanced targeting peptide displaywhen expressed in human exosome-producing cells (which remainsundetermined), utilizing human Lamp2b is desirable to increase theimmune compatibility of exosome-based therapeutics (24). Our strategyfor enhancing peptide display via human Lamp2b could also increasepeptide display via other exosomal membrane proteins because all suchpeptide fusions may be susceptible to degradation by endosomalproteases. Given the need for robust technologies for effectivelydirecting exosomes to traffic to specific destinations in vivo, thisstrategy for enhancing display of targeting peptides could generallyenable and enhance exosome-based therapeutics.

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In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

1-22. (canceled)
 23. A method of loading an exosome with a cargo nucleicacid, comprising expressing within an exosome-producing cell (i) afusion protein comprising a nucleic acid-binding domain and anexosome-targeting domain, and (ii) a cargo nucleic acid comprising abinding motif; wherein the binding motif of the cargo nucleic acid bindsto the nucleic acid-binding domain of the fusion protein and theexosome-targeting domain of the fusion protein localizes to an exosome,such that the cargo nucleic acid is loaded into the exosome when thefusion protein and cargo nucleic acid are both expressed by theexosome-producing cell.
 24. The method of claim 23, wherein the fusionprotein further comprises an engineered glycosylation site, wherein theengineered glycosylation site comprises an amino acid sequence that isnot naturally present in the fusion protein or any of the components ofthe fusion protein.
 25. The method of claim 23, wherein theexosome-targeting domain of the fusion protein is a domain of alysosome-associated protein.
 26. The method of claim 25, wherein thelysosome-associated protein is a lysosome-associated membrane protein(LAMP).
 27. The method of claim 26, wherein the LAMP comprises a luminalN-terminus and a cytoplasmic C-terminus.
 28. The method of claim 23,wherein the exosome-targeting domain of the fusion protein comprises asequence selected from a group consisting of SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.
 29. The method of claim23, wherein the exosome-targeting domain of the fusion protein comprisesa sequence having at least 80% amino acid sequence identity to asequence selected from a group consisting of SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.
 30. The method of claim23, wherein the nucleic acid-binding domain of the fusion proteincomprises a binding domain of a bacteriophage.
 31. The method of claim30, wherein the bacteriophage is a MS2 bacteriophage, a R17bacteriophage, a lambdoid bacteriophage, a P22 bacteriophage, or a phi21bacteriophage.
 32. The method of claim 26, wherein the nucleicacid-binding domain of the fusion protein comprises a binding domain ofa bacteriophage.
 33. The method of claim 32, wherein the bacteriophageis a MS2 bacteriophage, a R17 bacteriophage, a lambdoid bacteriophage, aP22 bacteriophage, or a phi21 bacteriophage.
 34. The method of claim 23,wherein the cargo nucleic acid is an RNA.
 35. The method of claim 34,wherein the RNA is a therapeutic RNA.
 36. The method of claim 34,wherein the binding motif comprises one or more high affinity bindingloops comprising a sequence and structure selected from the groupconsisting of:

where N—N is any two base-paired RNA nucleotides.
 37. The method ofclaim 23, wherein the exosome-targeting domain of the fusion proteincomprises a domain of a lysosome-associated membrane protein (LAMP), thenucleic acid-binding domain of the fusion protein comprises a bindingdomain of a bacteriophage, and the cargo nucleic acid is an RNA.
 38. Themethod of claim 23, wherein the exosome-targeting domain of the fusionprotein comprises a sequence selected from a group consisting of SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36; thenucleic acid-binding domain of the fusion protein comprises a bindingdomain of a bacteriophage selected from a group consisting of a MS2bacteriophage, a R17 bacteriophage, a lambdoid bacteriophage, a P22bacteriophage, and a phi21 bacteriophage; and the cargo nucleic acid isan RNA.
 39. The method of claim 38, wherein the RNA is a therapeuticRNA.
 40. The method of claim 38, wherein the binding motif comprises oneor more high affinity binding loops comprising a sequence and structureselected from the group consisting of:

where N—N is any two base-paired RNA nucleotides.